A goal of plant breeding is to combine, in a single plant, various desirable traits. For field crops such as corn, these traits can include greater yield and better agronomic quality. However, genetic loci that influence yield and agronomic quality are not always known, and even if known, their contributions to such traits are frequently unclear. Thus, new loci that can positively influence such desirable traits need to be identified and/or the abilities of known loci to do so need to be discovered.
Previous studies have focused primarily on the identification and manipulation of candidate genes that encode proteins, such as transcription factors. These genes could encode proteins that directly affect the physiology of the plant or transcription factors that regulate these effector genes.
miRNAs are post-transcriptional regulators that bind to complementary sequences of target messenger RNA transcripts, and there is evidence that they play an important role in regulating gene activity. These 20-22 nucleotide noncoding RNAs have the ability to hybridize via base pairing with specific target mRNAs and downregulate the expression of these transcripts by mediating either RNA cleavage or translational repression.
Numerous efforts are ongoing to discover miRNA genes that influence plant traits. These efforts rely on classic molecular biology cloning and expression techniques, as well as computational methods (see, e.g., U.S. Patent Application Publication No. 20070118918). miRNAs have already been shown to play important roles in plant development, signal transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis (Zhang et al. (2006) Dev. Biol. 289:3-16). Further, miRNAs have been shown to control a variety of plant developmental processes including flowering time, leaf morphology, organ polarity, floral morphology, and root development (reviewed by Mallory and Vaucheret (2006) Nat. Genet. 38:S31-36).
In general, plant miRNAs share a high degree of complementarity with their targets (reviewed by Bonnet et al. (2006) New Phytol. 171:451-468), and the predicted mRNA targets of plant miRNAs identified by computational methods encode a wide variety of proteins. Many of these proteins are transcription factors, which may have roles in development. Others are enzymes that have putative roles in mitochondrial metabolism, oxidative stress response, proteasome function, and lignification.
At least 30 miRNA families have been identified in Arabidopsis (reviewed by Meyers et al. (2006) Curr. Opin. Biotech. 17:1-8), and many of these miRNA sequences are associated with more than one locus, bringing the total number up to approximately 100. As the particular miRNAs identified by various investigators have not generally overlapped, it is assumed that the search for the entire set of miRNAs expressed by a given plant genome, the “miRNome,” is not yet complete. One reason for this might be that many miRNAs are expressed only under very specific conditions, and thus may have been missed by standard cloning efforts. A study by Sunkar and Zhu (2004, Plant Cell 1(6):2001-2019) suggests that, indeed, miRNA discovery may be facilitated by choosing “non-standard” growth conditions for library construction. Sunkar and Zhu identified novel miRNAs in a library consisting of a variety of stress-induced tissues and they demonstrated induction of some of these miRNAs by drought, cold and other stresses, suggesting a role for miRNAs in stress responses. This conclusion is reinforced by the observation that miRNA targeting genes in the sulfur assimilation pathway were shown to be induced under conditions of sulfate starvation (Jones-Rhoades and Bartel (2004) Mol. Cell. 14:787-799).
However, what has gone completely unappreciated up to this point is that polymorphisms present in miRNA regions (i.e., a region of a chromosome coding for a mature miRNA, pre-miRNA and flanking sequences) have a measurable impact on plant phenotype. Accordingly, using this knowledge a skilled artisan can manipulate plants and plant materials using both and classic molecular biology techniques and traditional breeding techniques to introduce desirable traits into plant varieties. For example, desirable loci can be introgressed into commercially available plant varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those progeny plants that contain one or more loci that encode the desired traits. Such identification and selection may be based on selection of informative markers that are associated with desired traits. MAB can also be used to develop near-isogenic lines (NIL) harboring loci of interest, allowing a more detailed study of the effect each locus has on a desired trait, and is also an effective method for development of backcross inbred line (BIL) populations.